ABSTRACT
BACKGROUND: Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells, which have been listed as the preferred stem cells for the treatment of spinal cord injury. However, due to their low differentiation efficiency, it is particularly important to find a factor with high induction ability. Based on literature review and our previous studies, it is speculated that bone morphogenetic protein 7 (BMP-7) gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method, and then were transfected with LV-GFP when multiplicities of infection were 50, 25, 10, and 1. Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection, to confirm the best multiplicity of infection. Passage 3 bone marrow mesenchymal stem cells were divided into blank control group (routine culture), LV-GFP group, and LV-BMP-7-GFP group, followed by transfection at the best multiplicity of infection. After 24, 48, 72, 96, and 120 hours of transfection, MTT assay was used to detect cell survival rate in each group. Immunocytochemical assay was used to detect the expression of nerve cell markers (neurofilament protein 200, synaptophysin-1) after 3 days of transfection. RESULTS AND CONCLUSION: (1) After 3 days of LV-GFP transfection, GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10, 25, and 50, whereas no GFP-positive cells were found when the multiplicity of infection was 1. The average fluorescence intensity was the highest when the multiplicity of infection was 10 (P < 0.05), indicating that multiplicity of infection=10 had the best infection effect. (2) Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group, but positive in the LV-BMP-7-GFP group. The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown. In summary, lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.
ABSTRACT
OBJECTIVE@#To observe the repair effect of bone marrow mesenchymal stem cells (BMSCs) combined with basic fibroblast growth factor (bFGF) on spinal cord injury in rats and explore its mechanism.@*METHODS@#SD rat BMSCs were obtained by serum culture technique. Eighty healthy 6-week-old male SD rats(weight about 240 g) were randomly divided into 4 groups with 20 each. The sham operation group underwent simple laminectomy without damaging spinal cord and was kept in the same condition as the other 3 groups. The other 3 groups underwent left T9 spinal cord hemisection to establish spinal cord injury model. After 9 days of modeling the local transplantation was performed. The Control group was implanted with gelatin sponge containing normal saline. The BMSCs transplantation group was implanted with gelatin sponge containing BMSCs. The bFGF+BMSCs transplantation group was implanted with gelatin sponge containing bFGF+BMSCs. After 4 and 8 weeks, the expression of NF-200 and GFAP in injured spinal cord tissue was analyzed by Western blotting and the recovery of hind limb function was evaluated by Basso Beattie Bresnahan(BBB) motor function score scale.@*RESULTS@#The BBB scores of BMSCs transplantation group and bFGF+BMSCs transplantation group were better than control group at 4 and 8 weeks after operation (<0.05) and there was significant difference between bFGF+BMSCs transplantation group and BMSCs transplantation group (<0.05). After 4 and 8 weeks postoperatively, NF-200 expression was minimal in control group and only a small amount was expressed in BMSCs transplantation group while in bFGF+BMSCs transplantation group NF-200 was highly expressed(<0.05). GFAP expression was high in control group, middle in BMSCs transplantation group and low in bFGF BMSCs transplantation group(<0.05). There was significant difference between bFGF+BMSCs transplantation group, BMSCs transplantation group and control group(<0.05).@*CONCLUSIONS@#The combined transplantation of BMSCs and bFGF can repair the spinal cord injury in rats. The mechanism may be related to the decrease of GFAP expression and the increase of NF-200 expression.
Subject(s)
Animals , Male , Rats , Bone Marrow Cells , Fibroblast Growth Factor 2 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Rats, Sprague-Dawley , Spinal Cord , Spinal Cord InjuriesABSTRACT
Objective To investigate the effects of quercetin on glial scar formation and axonal regeneration after spinal cord injury (SCI) and its association with the p38 mitogen activated protein kinase (MAPK) signal pathway.Methods 128 female Sprague-Dawley (SD) rats were randomly divided into a control group (SCI + saline),an intervention group (SCI + quercetin + anisomycin),a treatment group (SCI + quercetin) and a sham-operation group (n =32).Basso Beattie Bresnahan (BBB) assessment and footprint analysis of the hind limb were performed on days 1,3,7,14,21 and 28 postoperation in each group.The expression levels of p38MAPK,phosphorylation p38MAPK,glial fibrillary acidic protein (GFAP) and neurofilament protein-200 (NF-200) were detected by Western blot.The numbers of GFAP and NF-200 positive staining cells in the injured spinal cord in each group were detected by immunohistochemistry.Results The BBB scores in the treatment group were significantly higher than in the intervention and control groups at each time point after SCI except on day 3 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in each SCI group were significantly higher than in the sham-operation group on days 3 and 7 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in the treatment group were significantly lower than in the control and intervention groups on days 3,7 and 14 postoperation (P ＜ 0.05),but there was no significant difference on day 28 postoperation among all the groups (P ＞ 0.05).The numbers of NF-200 and GFAP positive staining cells were significantly greater than in the sham-operation group at each time point postoperation (P ＜ 0.05);the NF-200 positive staining cells in the treatment group were significantly increased in comparison with the control and intervention groups (P ＜ 0.05);the GFAP positive staining cells in the treatment group were significandy fewer than in the control and intervention groups on days 7,14 and 28 postoperation (P ＜ 0.05).Conclusions Quercetin may have protective effects against acute SCI by decreasing glial scar formation,increasing axonal regeneration and promoting recovery of locomotor and nerve function in rats.The effects may be correlated with inhibition of the p38MAPK signal pathway.